Abstract

People with spinal cord injury (SCI) have three- to four-fold greater risk of cardiovascular disease (CVD) compared with those without SCI. Although circulating extracellular microvesicles are key effectors of vascular health and disease, how their functional phenotype might be altered with SCI is unknown. The aim of the present study was to determine the effects of microvesicles isolated from SCI adults on endothelial cell inflammation and oxidative stress as well as endothelial nitric oxide (NO) synthase (eNOS) activation and tissue-type plasminogen activator (t-PA) expression. Eighteen young and middle-aged adults were studied: 10 uninjured (7M/3F; age: 39 ± 3 years) and 8 cervical level spinal cord injured (SCI; 7M/1F; 46 ± 4 years; cervical injury: C3: n=1; C5: n=4; C6: n=3). Circulating microvesicles were isolated, enumerated and collected from plasma by flow cytometry. Human umbilical vein endothelial cells (HUVECs) were cultured and treated with microvesicles from either the uninjured or SCI adults. Microvesicles from SCI adults did not affect cellular markers or mediators of inflammation and oxidative stress. However, microvesicles from the SCI adults significantly blunted eNOS activation, NO bioavailability and t-PA production. Intercellular expression of phosphorylated eNOS at Ser1177 and Thr495 sites, specifically, were ∼65% lower and ∼85% higher, respectively, in cells treated with microvesicles from SCI compared with uninjured adults. Decreased eNOS activity and NO production as well as impaired t-PA bioavailability renders the vascular endothelium highly susceptible to atherosclerosis and thrombosis. Thus, circulating microvesicles may contribute to the increased risk of vascular disease and thrombotic events associated with SCI.

Introduction

Permanent spinal cord injury (SCI) is associated with an increased risk and prevalence of cardiovascular disease (CVD)-related morbidity and mortality [1,2]. Atherosclerosis, coronary heart disease and stroke are among the most common causes of death in individuals with chronic SCI [1–4]. The mechanisms and factors underlying the heightened, often accelerated, development of atherosclerotic CVD (and its clinical consequences) with chronic SCI are complex, multifactorial and not completely understood [3,4]. While there is a general increase in the preponderance of CVD risk factors such as, physical inactivity, obesity, blood pressure instability, dyslipidemia and metabolic syndrome in SCI individuals; risk factor burden does not fully account for the increased vascular disease risk with SCI. Indeed, the risk of heart disease and stroke has been reported to be several-fold higher in adults with SCI compared with those without SCI, even after controlling for traditional CVD risk factors [4]. Thus, other factors are likely involved in perturbing the vascular system leading to a proatherogenic environment [4]. For example, elevations in markers of endothelial cell activation [5] and dysfunction [6] have been reported in adults with SCI; endothelial activation and dysfunction are central factors in the etiology of atherosclerosis [7,8]. Mediating factors involved with SCI-related vascular dysfunction and disease, however, are not well understood.

Once thought to be inert cellular debris, circulating extracellular microvesicles are now recognized as potent vectors involved in the transcellular exchange of biological signals (both physiologic and pathologic) and cell-to-cell communication [9]. Microvesicles are small (100–1000 nm) aneucliod membrane vesicles that are released into the circulation by various cell types (e.g. endothelial cells, platelets, leukocytes, monocytes) in response to a myriad of physiologic and pathologic processes including: exposure to high shear stress, cellular activation stemming from proinflammatory, prothrombotic and/or proapoptotic stimuli, cellular differentiation, cell senescence and apoptosis [10,11]. The vascular endothelium is a central and key target of circulating microvesicles. Dependent upon the stimulus and physiological or pathological condition governing their release, microvesicles can induce a proatherosclerotic endothelial phenotype by triggering endothelial inflammation [12,13] and oxidative stress as well as impairing nitric oxide (NO) production [14–16]. Currently, it is unknown whether SCI influences the functional phenotype of circulating microvesicles.

Accordingly, the experimental aim of the present study was to determine the effects of microvesicles isolated from SCI adults on endothelial cell inflammation and oxidative stress as well as endothelial NO synthase (eNOS) activation and tissue-type plasminogen activator (t-PA) expression. These key cellular indicators of endothelial cell function are known to play pivotal roles in vascular health and disease [8]. Currently, it is unknown whether SCI is associated with an altered functional phenotype of circulating microvesicles.

Materials and methods

Subjects

Eighteen young and middle-aged adults (27–64 years) were studied: 10 uninjured (7M/3F) and 8 chronic cervical level spinal cord injured (SCI; 7M/1F; cervical injury: C3: n=1; C5: n=4; C6: n=3). The SCI subjects were screened in accordance with the American Spinal Injury Association’s (ASIA) Impairment Scale (AIS; A: n=1; B: n=4; C: n=2; D: n=1). The average time since injury for the SCI adults was 26 ± 5 years. All subjects were normotensive and free of overt cardiovascular and metabolic disease assessed by medical history, resting electrocardiograms and fasting blood chemistry. Prior to participation in the study, all subjects had the research study and its potential risks and benefits explained before providing written informed consent according the Ethics Committee of the School of Medicine at the University of Split, Croatia. The study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki. The present study was a smaller part of a larger investigation [17]; however, although some of the same volunteers are included, the molecular and biochemical data presented herein addresses novel, de novo experimental aims.

Body composition and metabolic measures

Body mass was measured to the nearest 0.1 kg. Body mass index (BMI) was calculated as weight (kg) divided by height (m) squared. Fasting plasma lipid, lipoprotein, glucose and insulin concentrations were determined using standard techniques.

Circulating markers of inflammation and oxidative stress

Plasma concentrations of interleukin (IL)-6 and tumor necrosis factor (TNF)-α as well as oxidized-LDL (oxLDL) were determined by ELISA (R&D Systems, Minneapolis, MN; Mercodia, Winston Salem, NC). The sensitivity range for each assay is as follows: IL-6: 0.05–0.35 pg/ml; TNF-α: 0.481–2.24 pg/ml; oxLDL: 3.9 U/l (lowest detectable concentration). Intra-assay coefficient of variation for each assay was <10%.

Microvesicle identification and isolation

Microvesicle isolation was performed as previously described [16]. Briefly, venous blood was collected in sodium citrate tubes and centrifuged at 1500g for 10 min at room temperature; plasma was collected and stored at −80°C for batch analysis and microvesicle isolation. To isolate microvesicles from each subject sample for use in cell experiments, plasma samples were centrifuged at 13000g for 2 min to remove cellular debris and then re-centrifuged at 20500g for 30 min at 4°C to pellet microvesicles. The pelleted microvesicles were then re-suspended in media. To determine the concentration of the isolated microvesicles, media samples were centrifuged at 13000g for 2 min and 100 µl was transferred to a TruCount tube (BD Biosciences, Franklin Lakes, NJ, U.S.A.). Thereafter, samples were fixed with 2% paraformaldehyde (ChemCruz Biochemicals, Santa Cruz, CA, U.S.A.) and diluted with PBS. All samples were analyzed using an FACS Aria I flow cytometer (BD Biosciences). Microvesicle size threshold was established using Megamix-Plus SSC calibrator beads (Biocytex, Marseille, France) and only events >0.16 µm and <1 µm in size were counted. The concentration of microvesicles was determined using the formula: (number of events in region containing microvesicles/number of events in absolute count bead region) × (total number of beads per test/total volume of sample).

Microvesicle and endothelial cell culture

Human umbilical vein endothelial cells (HUVECs) (Life Technologies, Thermo Fisher, Waltham, MA, U.S.A.) were cultured in endothelial growth media (EBM-2, BulletKit) (Lonza, Basel, Switzerland) supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin under standard cell culture conditions (37°C and 5% CO2). Growth media was replaced 24 h after initial culture and every 2 days thereafter. Cells were serially passaged after reaching 80-90% confluence and harvested for experimentation after reaching ∼90% confluence on the third passage. HUVECs were seeded into six-well tissue culture plates with media containing an equal concentration of microvesicles from either an uninjured or SCI adult for 24 h. Cells were treated with microvesicles on a 2:1 (microvesicle:endothelial cell) ratio [16]. After treatment, cells and media were harvested for the determination of cellular protein expression, miRNA expression and soluble cytokine release.

Endothelial cell cytokine, NO and t-PA release

Concentrations of IL-6, IL-8, TNF-α and t-PA in the supernatant from microvesicle-treated cells were determined using chemiluminescent ELISA (R&D Systems, Minneapolis, MN; Elabscience, Houston, TX). To assess NO production total nitrite in the supernatant was determined using the Total Nitric Oxide and Nitrate/Nitrite Paramaeter Assay Kit (R&D Systems, Minneapolis, MN). The sensitivity range for the NO and t-PA assay are as follows: NO: 0.09–0.78 µmol/l; t-PA: 75 pg/ml (lowest detectable concentration). Intra-assay coefficient of variation for the media-based ELISAs were <10% for each assay.

Endothelial cell protein expression

Whole cell lysates were obtained from microvesicle-treated HUVECs for the quantification of intracellular proteins as previously described [16]. Microvesicle-treated HUVECs were washed in ice-cold PBS and incubated in ice-cold RIPA buffer containing protease and phosphatase inhibitors (Thermo Fisher) for 10 min. Cell lysates were sonicated for 20 s (four 5-s cycles spaced by 90 s between each cycle), incubated on ice for an additional 20 min and then centrifuged at 13000g at 4°C for 10 min. Following centrifugation, the supernatant was collected and protein concentration was determined using the Bio-Rad DC protein assay (Bio-Rad, Hercules, CA). Protein expression was measured by capillary electrophoresis immunoassay (Wes, ProteinSimple, San Clara, CA). Briefly, 2–3 ng of cell lysate was combined with a provided sample master mix (ProteinSimple, Santa Clara, CA) containing of 1× sample buffer, 1× fluorescent molecular weight markers and 40 mM DTT. Samples were vortexed and heated at 95°C for 5 min prior to combining with blocking solution, primary antibodies, horseradish peroxidase–conjugated secondary antibody, chemiluminescent substrate, and separation and stacking matrices for automated electrophoresis (375 V for 25 min) and immunodetection using the Wes system [16]. Protein expression was normalized to total protein in the sample and presented as arbitrary units (AUs). Rabbit primary antibodies against nuclear factor-κB (NF-κB) p65 (D14E12), phospho (p)-NF-κB p65 (Ser536) (93H1), (diluted 1:50 and 1:25, respectively) (Cell Signaling Technology, Danvers, MA); eNOS (#PA1-037), p-eNOS (Ser1177) (#PA5-35879), p-eNOS (Ser633) (#PA5-64551), p-eNOS (Thr495) (#9574S), superoxide dismutase (SOD)1 (#2770), catalase (D5N7V)(#14097) and t-PA (#PA5-51927) (diluted 1:25, 1:25, 1:25; 1:25; 1:25; 1:25 and 1:10, respectively) (Thermo Fisher, Waltham, MA) and heat shock protein (Hsp) 70 (NBPI-77456) (diluted 1:25) (Novus Biolgicals, Littleton, CO) were used. Initial titrations were performed to optimize antibody and total protein concentration for each protein.

Endothelial cell oxidative stress

In addition to cellular expression of SOD and catalase, cellular reactive oxygen species (ROS) was also determined. HUVECs were seeded in 96-well tissue culture plates (Thermo Scientific, Waltham, MA, U.S.A.) and allowed to adhere for at least 1 h. Adherent cells were then treated with media or media containing microvesicles for 3 h. Cells were then treated with CellROX® Deep Red reagent (Life Technologies, Carlsbad, CA, U.S.A.) at a final concentration of 5 μM for 30 min. Immediately thereafter, the cells were washed three times with PBS and fluorescence was measured using a GEMINI EM microplate reader (Molecular Devices, Sunnyvale, CA) and reported as the percentage relative to control [18].

Endothelial cell miRNA expression

miRs associated with cellular inflammation (mir-126, miR-146a and miR-181b), oxidative stress (miR-200c) and eNOS (miR-21 and miR-126) were determined by RT-PCR [19]. After microvesicle treatment, 1.0 × 105 cells were collected and total cellular RNA was isolated using the miRVANA RNA isolation kit (Exiqon, Vedbake, Denmark). RNA concentration was determined using a Nanodrop Lite spectrophotometer (Thermo Fisher, Waltham, MA). Thereafter, 150 ng of RNA was reverse transcribed using the miScript II Reverse Transcription Kit (Qiagen, Hilden, Germany). RT-PCR was performed using Bio-Rad CFX96 RT-PCR with the miScript SYBR green PCR kit (Qiagen, Hilden, Germany) and primers for miR-21, miR-126, miR-146a, miR-181b, miR-200c and U6 (Qiagen, Hilden, Germany). All samples were assayed in duplicate. miRNA expression was quantified using the comparative Ct method and normalized to U6 [19]. Relative expression of each transcript was calculated as the 2−ΔCt where 2−((Ct[miR] − Ct[RNU6]) and presented as AU.

Statistical analysis

The distribution of the data was assessed by the Shapiro–Wilk test and the homogeneity of variances by the Levene test. Group differences in subject characteristics, circulating inflammation and oxidative stress markers, cellular protein miRNA expression as well as cellular oxidative stress were determined by either analysis of variance (normally distributed data) or Mann–Whitney U test (non-normally distributed data). Data are presented as mean ± SEM for normally distributed variables and as the median (interquartile range) for non-normally distributed variables. Pearson correlations were determined between variables of interest. Statistical significance was set a priori at P<0.05.

Results

Subject characteristics are presented in the Table 1. There were no significant group differences in age, anthropometric or hemodynamic measurements. Although total cholesterol was slightly, albeit significantly, higher in the SCI subjects, there were no significant group differences in other metabolic variables. In addition, plasma concentrations of IL-6, TNF-α and oxLDL were not significantly different between the uninjured and SCI adults (Table 1).

Table 1
Select subject characteristics
VariableUninjured (n=10)Spinal Cord Injured (n=8)
Age (years) 39 ± 3 46 ± 4 
Body mass (kg) 76.5 ± 3.6 79.3 ± 3.7 
BMI (kg/m224.0 ± 0.7 23.3 ± 1.1 
Systolic BP (mmHg) 117 ± 6 117 ± 3 
Diastolic BP (mmHg) 68 ± 3 72 ± 4 
Total cholesterol (mg/dl) 131.0 ± 8.0 157.0 ± 9.3* 
LDL-cholesterol (mg/dl) 78.0 ± 4.4 88.0 ± 7.6 
HDL-cholesterol (mg/dl) 41.8 ± 3.0 45.9 ± 4.8 
Triglycerides (mg/dl) 75.7 ± 8.6 106.4 ± 16.2 
Glucose (mg/dl) 89.6 ± 6.7 86.5 ± 3.6 
Insulin (µU/ml) 8.6 ± 2.1 9.9 ± 3.0 
IL-6 (pg/ml) 1.3 ± 0.2 1.7 ± 0.2 
TNF-α (pg/ml) 4.8 ± 0.2 5.4 ± 0.3 
Ox-LDL (U/l) 51.2 ± 3.3 50.8 ± 4.8 
VariableUninjured (n=10)Spinal Cord Injured (n=8)
Age (years) 39 ± 3 46 ± 4 
Body mass (kg) 76.5 ± 3.6 79.3 ± 3.7 
BMI (kg/m224.0 ± 0.7 23.3 ± 1.1 
Systolic BP (mmHg) 117 ± 6 117 ± 3 
Diastolic BP (mmHg) 68 ± 3 72 ± 4 
Total cholesterol (mg/dl) 131.0 ± 8.0 157.0 ± 9.3* 
LDL-cholesterol (mg/dl) 78.0 ± 4.4 88.0 ± 7.6 
HDL-cholesterol (mg/dl) 41.8 ± 3.0 45.9 ± 4.8 
Triglycerides (mg/dl) 75.7 ± 8.6 106.4 ± 16.2 
Glucose (mg/dl) 89.6 ± 6.7 86.5 ± 3.6 
Insulin (µU/ml) 8.6 ± 2.1 9.9 ± 3.0 
IL-6 (pg/ml) 1.3 ± 0.2 1.7 ± 0.2 
TNF-α (pg/ml) 4.8 ± 0.2 5.4 ± 0.3 
Ox-LDL (U/l) 51.2 ± 3.3 50.8 ± 4.8 

Values are mean ± SEM. Abbreviations: BP, blood pressure; HDL, high-density lipoprotein; LDL, low-density lipoprotein.

*P<0.05 vs. Uninjured.

Endothelial cell inflammation

The effect of microvesicles on cellular markers and mediators of inflammation is shown in Figure 1. There was no significant difference in microvesicle-induced endothelial release of IL-6 (mean ± SEM: 36.5 ± 1.5 vs 36.5 ± 0.8 pg/ml; Figure 1A), IL-8 (mean ± SEM: 44.7 ± 1.8 vs 45.8 ± 2.2 pg/ml; Figure 1B) and TNF-α (mean ± SEM: 3.7 ± 0.1 vs 3.6 ± 0.1 pg/ml; Figure 1C) between microvesicles from the uninjured and SCI adults. In addition, cell expression of NF-κB p65 (mean ± SEM: 90.0 ± 5.5 vs 81.6 ± 6.6 AU; Figure 1D) and p-NF-κB p65 (Ser536; active NF-κB) (mean ± SEM: 82.2 ± 18.5 vs 95.1 ± 22.2 AU; Figure 1E), miR-146a (median [IQR]: 1.6 [0.7–2.0] vs 1.3 [0.4–1.8] AU; Figure 1F) and miR-181b (median [IQR]: 1.7 [1.2–2.3] vs 1.3 [0.7–1.9] AU; Figure 1G) was not significantly different in HUVECs treated with microvesicles from uninjured compared with SCI adults.

Effects of Microvesicles on Markers and Mediators of Endothelial Cell Inflammation

Figure 1
Effects of Microvesicles on Markers and Mediators of Endothelial Cell Inflammation

Endothelial cell release of IL-6 (A), IL-8 (B), TNF-α (C) and intracellular expression of NF-κB p65 (D), p-NF-κB p65 (Ser536) (E), miR-146a (F) and miR-181b (G) in response to treatment with microvesicles from uninjured and SCI adults. Mean value is denoted for IL-6, IL-8, TNF-α and NF-κB data; median value for miR-146a and miR-181b.

Figure 1
Effects of Microvesicles on Markers and Mediators of Endothelial Cell Inflammation

Endothelial cell release of IL-6 (A), IL-8 (B), TNF-α (C) and intracellular expression of NF-κB p65 (D), p-NF-κB p65 (Ser536) (E), miR-146a (F) and miR-181b (G) in response to treatment with microvesicles from uninjured and SCI adults. Mean value is denoted for IL-6, IL-8, TNF-α and NF-κB data; median value for miR-146a and miR-181b.

Endothelial cell oxidative stress

ROS production was similar in HUVECs treated with microvesicles from uninjured compared with spinal cord injured adults (median [IQR]: 62 [56–78] vs 69 [56–82]% of control; Figure 2A). In addition, cellular expression of SOD1 (mean + SEM: 389.2 ± 57.2 vs 376.4 ± 79.9 AU; Figure 2B) and catalase (mean + SEM: 964.0 ± 101.3 vs 983.5 ± 213.6 AU; Figure 2C) was not significantly different between cells treated with microvesicles from uninjured compared with spinal cord injured adults. The expression of the ROS-induced chaperone protein, Hsp70 (mean ± SEM: 69.9 ± 2.5 vs 66.7 ± 5.1 AU; Figure 2D) and miR-200c (mean ± SEM: 1.3 ± 0.1 vs 1.2 ± 0.4 AU; Figure 2E) was also not significantly different in HUVECs treated with microvesicles from uninjured compared with spinal cord injured adults.

Effects of Microvesicles on Markers and Mediators of Endothelial Cell Oxidative Stress

Figure 2
Effects of Microvesicles on Markers and Mediators of Endothelial Cell Oxidative Stress

Endothelial cell ROS production (A), SOD1 (B), catalase (C), intracellular Hsp70 (D) and miR-200c (E) expression in response to treatment with microvesicles from uninjured and SCI adults. Mean value is denoted for SOD1, catalase and Hsp70; median value for ROS and miR-200c.

Figure 2
Effects of Microvesicles on Markers and Mediators of Endothelial Cell Oxidative Stress

Endothelial cell ROS production (A), SOD1 (B), catalase (C), intracellular Hsp70 (D) and miR-200c (E) expression in response to treatment with microvesicles from uninjured and SCI adults. Mean value is denoted for SOD1, catalase and Hsp70; median value for ROS and miR-200c.

eNOS and t-PA expression

Cellular expression of p-eNOS (Ser1177), p-eNOS (Ser633), p-eNOS (Thr495), NO production and miR-21 and miR-126 are shown in Figure 3. Total eNOS (85.4 ± 8.0 vs 85.7 ± 3.4 AU) and p-eNOS (Ser633) (171.0 ± 26.9 vs 143.3 ± 26.1 AU; Figure 3B) expression was not significantly different between cells treated with microvesicles from uninjured and SCI adults. However, p-eNOS (Ser1177) (mean ± SEM: 7.1 ± 1.7 vs 13.4 ± 1.4 AU; Figure 3A) expression was significantly lower (∼65%) and p-eNOS (Thr495) (mean ± SEM: 102.6 ± 16.7 vs 55.2 ± 7.9 AU; Figure 3C) significantly higher (∼85%) in cells treated with microvesicles from SCI adults compared with microvesicles from uninjured adults. Concordantly, NO production was significantly lower (∼30%; 3.9 + 0.5 vs 5.6 + 0.5 µmol/l; Figure 3D) in cells treated with microvesicles from SCI adults. Cellular expression of miR-21 (median [IQR]: 1.7 [1.1–3.6] vs 9.8 [1.6–13.9] AU; Figure 3E) and miR-126 (mean ±SEM: 3.8 ± 0.9 vs 9.7 ± 2.1 AU; Figure 3F) were also significantly lower (∼75 and ∼60%, respectively) in cells treated with microvesicles from SCI adults compared with uninjured adults.

Effects of Microvesicles on Markers and Mediators of Endothelial Cell Nitric Oxide Prodution

Figure 3
Effects of Microvesicles on Markers and Mediators of Endothelial Cell Nitric Oxide Prodution

Endothelial cell expression of p-eNOS (Ser1177) (A), p-eNOS (Ser633) (B), p-eNOS (Thr495) (C), NO production (D), miR-21 (E) and miR-126 (F) in response to treatment with microvesicles from uninjured and SCI adults. Mean value is denoted for p-eNOS, NO production and miR-126; median value for miR-21. *P<0.05 vs uninjured.

Figure 3
Effects of Microvesicles on Markers and Mediators of Endothelial Cell Nitric Oxide Prodution

Endothelial cell expression of p-eNOS (Ser1177) (A), p-eNOS (Ser633) (B), p-eNOS (Thr495) (C), NO production (D), miR-21 (E) and miR-126 (F) in response to treatment with microvesicles from uninjured and SCI adults. Mean value is denoted for p-eNOS, NO production and miR-126; median value for miR-21. *P<0.05 vs uninjured.

Endothelial cell expression of t-PA and t-PA antigen release is shown in Figure 4. Both t-PA expression (median [IQR]: 27.9 [25.6–40.7] vs 47.7 [25.7–40.7] AU; Figure 4A) and amount of t-PA antigen (mean ± SEM: 131 ± 2 vs 161 ± 7 ng/ml; Figure 4B) released into the media were significantly lower (∼35 and ∼20%, respectively) in the HUVECs treated with microvesicles from SCI compared with uninjured adults.

Effects of Microvesicles on Endothelial Cell t-PA Production

Figure 4
Effects of Microvesicles on Endothelial Cell t-PA Production

Endothelial cell expression of t-PA (A) and t-PA antigen (B) released into the media in response to treatment with microvesicles from uninjured and SCI adults. Median value is denoted for t-PA expression. Mean value is denoted for t-PA antigen. *P<0.05 vs uninjured.

Figure 4
Effects of Microvesicles on Endothelial Cell t-PA Production

Endothelial cell expression of t-PA (A) and t-PA antigen (B) released into the media in response to treatment with microvesicles from uninjured and SCI adults. Median value is denoted for t-PA expression. Mean value is denoted for t-PA antigen. *P<0.05 vs uninjured.

Discussion

The seminal finding of the present study is that microvesicles isolated from SCI adults reduce eNOS activation, NO production and t-PA expression. Diminished eNOS activity, NO bioavailaility and t-PA production are known to render the vascular endothelium highly susceptible to atherosclerotic and thrombogenic processes [20,21]. Considering that accelerated atherosclerosis and thrombosis are major causes of morbidity and mortality in adults with SCI [3], circulating microvesicle phenotype may be a contributing factor.

The activation of eNOS and, in turn, NO production play an essential role in the regulation of endothelial function and vascular health [22]. eNOS is the product of the NOS3 gene that is expressed constitutively in endothelial cells [23]. Both animal and clinical studies have demonstrated the importance of eNOS in regulating vascular tone, suppressing vascular smooth muscle proliferation, inhibiting platelet aggregation and preventing leukocyte adhesion to the vessel wall [21,24]. Consequently, reduced eNOS activation is considered to be a major factor underlying endothelial cell dysfunction and an initiating event in the development of atherosclerosis, ischemia and thrombosis [21]. In experimental models of SCI, expression of eNOS RNA and activated eNOS has been shown to significantly decline in spinal cord tissue in response to injury [25], potentially contributing to the profound and rapid onset of post-traumatic ischemia in the affected spinal cord region [26]. In the present study, circulating microvesicles harvested from adults with SCI significantly reduced eNOS activation in endothelial cells. Phospohorylation is the primary post-translational modification regulating eNOS enzyme activity [27]. The expression of p-eNOS (Ser1177) was ∼65% lower and p-eNOS (Thr495) was ∼85% higher in HUVECs treated with microvesicles from SCI compared with uninjured adults. Phosphorylation of Ser1177 confers the greatest activation of eNOS; conversely phosphorylation of Thr495 attenuates eNOS activity [27–29]. Thus, microvesicles from spinal cord injured adults adversely affects eNOS activation by affecting the phosphorylation of two major regulatory sites involved in activation and inhibition of the enzyme. Consistent with the observed differences in p-eNOS (Ser1177) and p-NOS (Thr495), NO production was markedly lower in HUVECs treated with microvesicles from SCI adults, demonstrating the consequential effect of impairing eNOS activation. Of note, eNOS protein expression was not affected by the SCI-microvesicles indicating that the lower eNOS activation observed in response to SCI-microvesicles was not a consequence of lower protein expression, but rather suppressed activation. In this regard, changes in the cellular expression of miR-21 and miR-126, key miRNAs that directly influence eNOS activation, provide some mechanistic insight regarding the adverse effect of SCI-related microvesicles on eNOS. Concordant with a reduction in eNOS activation and NO production, the expression of miR-21 and miR-126 were significantly depressed in the cells treated with microvesicles from the SCI compared with uninjured adults. Both miR-21 and miR-126 are involved in the regulation of pathways that govern eNOS activation and function [30,31]. miR-21 suppresses phosphatase and tensin homolog (PTEN), a potent negative regulator of eNOS activation [30]. Unrestricted PTEN action has been shown to significantly blunt eNOS activity [32]. On the other hand, miR-126 safeguards the activity of the phosphatidylinositol-3 kinase/protein kinase B/eNOS signaling pathway and, in turn the potential for eNOS activation [31]. Although the expression patterns of miR-21 and miR-126 and eNOS activation provide etiological support for the involvement of these miRNAs in the SCI-microvesicle induced alteration in eNOS activation and NO production, we recognize that miRNA knockout studies are needed to fully characterize their mechanistic involvement.

From a clinical perspective, it is important to note that previous studies in other disease states have also reported adverse effects of circulating microvesicles on eNOS activity [16,33]. For example, microvesicles isolated from adults with coronary artery disease suppress NO-mediated endothelium-dependent relaxation in isolated arteries [33], providing in vitro mechanistic support for a clinical link between circulating microvesicles and disease severity and outcome. Thus, circulating microvesicle-induced blunting of eNOS activation and, in turn NO production, may contribute to SCI-related vascular dysfunction and disease risk.

In addition to reducing eNOS activation, SCI-related microvesicles also negatively affected endothelial cell t-PA protein expression and release. The ability of endothelial cells to synthesize and release t-PA is a critical endogenous defense mechanism against intravascular fibrin deposition and thrombosis [34]. Produced primarily by endothelial cells, t-PA is the key enzyme in initiating fibrinolysis by preferentially activating plasminogen on the surface of a developing thrombus [35]. Thus, endothelial t-PA production and release dictates the efficacy of endogenous fibrinolysis. Indeed, the thrombolytic importance of t-PA has been demonstrated in both animal and human studies showing that diminished ability of the endothelium to produce and release t-PA is an important antecedent to, and predictor of, atherothrombotic disease and events [34,36]. Impaired endothelial t-PA production and release is a common characteristic underlying the increased risk of atherothrombotic events associated with essentially all major CVD risk factors (e.g., aging, hypertension, obesity and dyslipidemia) [37–40]. HUVECs treated with microvesicles from the SCI adults exhibited markedly lower t-PA protein expression and release than cells treated with microvesicles from uninjured adults. Given the thrombolytic importance of t-PA, microvesicle-induced reduction in endothelial t-PA production may contribute to the increased risk of thrombosis and thrombotic events observed in chronic SCI adults [41–43].

In contrast with eNOS and t-PA, circulating microvesicles from the SCI adults did not affect cellular markers or mediators of inflammation or oxidative stress. Endothelial cell release of IL-6, IL-8 and TNF-α was not different between cells treated with microvesicles from the uninjured compared with SCI adults. Consistent with the cytokine response, cellular NF-κB expression and activation was not markedly altered by the microvesicles from the SCI adults. NF-κB is a primary transcription factor involved in regulating IL-6, IL-8 and TNF-α expression and release [44,45]. Further, intracellular expression of miR-146a and miR-181b, miRNAs directly involved in the regulation of NF-κB expression and activation, were not different in cells treated with microvesicles from the SCI compared with uninjured adults. Thus, taken together, circulating microvesicles from SCI adults do not induce a proinflammatory endothelial phenotype. This finding was somewhat unexpected considering the often-reported association between SCI and systemic inflammation [3,5]. However, the general health status of the SCI adults likely contributed to this finding. The SCI cohort presented no overt or clinical signs of acute or chronic inflammation. Chronic inflammatory burden in adults with chronic SCI is largely due to persistent, recurring conditions such as urinary tract infections and bed sores [46]. There was no evidence of either condition in our SCI group. Moreover, circulating concentrations of IL-6 and TNF-α were not elevated in the SCI adults, indicating the absence of systemic inflammatory stress. Similarly, circulating oxLDL was not elevated in the SCI adults suggestive of minimal systemic oxidative stress burden and, in turn, their microvesicles did not elicit a pro-oxidative endothelial phenotype.

The mechanisms responsible for the SCI-related microvesicle induced reduction in eNOS activation and t-PA expression are not clear. Increased cell inflammation and oxidative stress factors known to impair both eNOS activation and t-PA production, do not appear be contributing factors. Future studies are needed to determine the microvesicle RNA and protein cargo and its role in dictating the effects of microvesicles from SCI adults on eNOS, NO production and t-PA. The ability of microvesicles to transport, bind and transfer their content to recipient cells is a key factor underlying their cellular effects [47]. The composition of the microvesicle cargo can dramatically affect gene expression and, in turn, induce specific or sweeping phenotypic changes in target cells [48].

There are a few experimental considerations regarding the present study that deserve mention. First, with any cross-sectional human study there exists the possibility that genetic and/or lifestyle behaviors may have influenced the results of our group comparisons. In the present study, all SCI adults were tetraplegics and there were no differences between groups in adiposity, risk factor profile or cardiometabolic disease, thus limiting the influence of factors secondary to SCI on our results. Second, while the inclusion of only SCI adults with cervical level injury reduced variability and provided experimental specificity, it is nevertheless equally important to recognize that the circulating microvesicle phenotype may differ dependent upon the location and degree of SCI. Third, although we harvested microvesicles from a clinical population, the in vitro nature of our studies precludes us from making definitive translational statements regarding clinical risk. However, it is important to note that changes in eNOS activation and endothelial t-PA production under various experimental conditions in vitro have provided mechanistic insight for the link between both eNOS activity and t-PA bioavailability and cardiovascular risk and outcomes [34,49,50]. Fourth, our study population involved mainly men. Due to sample size, we were unable to address possible sex-related differences in SCI-microvesicle function; as such we cannot, and should not, automatically assume similar functional microvesicle phenotypes in women with SCI. Future studies are needed to address this important question. Finally, our in vitro studies involved the use of HUVECs and not an arterial-derived cell line; raising a potential concern that the expression of proteins and the function of venous endothelial cells in culture may not be representative of arterial endothelial cells and, in turn, may have limited impact regarding arterial atherosclerotic disease. However, several studies have demonstrated that the expression of key proteins involved in the regulation of vascular endothelial function and disease risk (such as: NF-κB and eNOS) is similar in endothelial cells acquired from an artery and vein in humans [51]. Moreover, venous and arterial cells have been shown to exhibit comparable, reproducible responses to various stimuli (physiologic and pathologic) both in vitro and in vivo [52–54]. Thus, cultured HUVECs are a reliable and often used in vitro cell line for studying endothelial cell function and atherogenic processes [54].

In conclusion, the results of the present study demonstrate that circulating microvesicles from adults with chronic cervical SCI reduce endothelial cell eNOS activation, NO production and t-PA bioavailability. Decreased eNOS activity and NO synthesis as well as reduced t-PA bioavailability are hallmark-independent features of an atheroprone vascular endothelium. Circulating microvesicles represent novel mechanistic mediators of the increased risk of vascular disease and thrombotic events associated with SCI.

Clinical perspectives

  • The risk and prevalence of CVD and atherothrombotic events is several-fold higher in adults with permanent SCI compared with uninjured adults. The mechanisms underlying the increased burden of atherosclerotic CVD with SCI are poorly understood and extend beyond traditional risk factors.

  • Circulating extracellular microvesicles are now recognized to play an important role in vascular health and disease. Microvesicles from SCI adults significantly reduce endothelial cell eNOS activation and t-PA production.

  • Decreased eNOS activity, NO production and impaired t-PA bioavailability are known contributors to increased atherothrombotic risk and events. Circulating microvesicles represent novel mechanistic mediators of the increased vascular risk associated with SCI.

Competing Interests

The authors declare that there are no competing interests associated with the manuscript.

Funding

This work was supported, at least in part, by the Seed Grant from the ICORD supported by the Blusson Integrated Cures Partnership (to P.N.A.); the Canada Research Chairs Program (to P.N.A.); the University of Calgary Start-up Funds (to A.A.P.); and the National Institutes of Health [grant numbers HL077450, HL107715 (to C.A.D.)].

Author Contribution

Conception and design of the work: L.M.B., G.B.C., O.F.B., T.M., Z.D., A.A.P., P.N.A. and C.A.D. Data acquisition, analysis and interpretation: L.M.B., G.B.C., V.P.G., J.G.H., N.M.D., K.A.S., J.J.G., P.N.A. and C.A.D. All authors contributed to the drafting of the manuscript for accuracy and critically important content. All authors approved the final version of the manuscript for submission for publication. All persons designated as authors qualify for authorship, and all those who qualify for authorship are listed.

Acknowledgements

We thank all the subjects who participated in the study as well as the staff at the University of Colorado Anschutz Medical Campus ACI/ID Flow Core for their technical assistance.

Abbreviations

     
  • AU

    arbitrary unit

  •  
  • CVD

    cardiovascular disease

  •  
  • eNOS

    endothelial nitric oxide synthase

  •  
  • Hsp

    heat shock protein

  •  
  • HUVEC

    human umbilical vein endothelial cell

  •  
  • IL

    interleukin

  •  
  • NF-κB

    nuclear factor-κB

  •  
  • NO

    nitric oxide

  •  
  • oxLDL

    oxidized-LDL

  •  
  • PTEN

    phosphatase and tensin homolog

  •  
  • ROS

    reactive oxygen species

  •  
  • SCI

    spinal cord injury

  •  
  • SOD

    superoxide dismutase

  •  
  • TNF

    tumor necrosis factor

  •  
  • t-PA

    tissue-type plasminogen activator

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